1. Field of the Invention
The present invention relates to a C-terminal .alpha.-amidating enzyme of porcine origin and a process for the production thereof.
2. Related Art
Recently it was found that various peptides and proteins isolated from the nerves and endocine systems have a C-terminal amino acid, the .alpha.-carboxyl group of which is amidated (--CONH.sub.2), and that this structure is often essential for physiological activities of the peptides and proteins. A general biosynthesis mechanism of such amidated peptides is understood to be that in which RNA is translated to a precursor of an amidated peptide, which is then amidated at the .alpha.-position of its C-terminal by a C-terminal .alpha.-amidating enzyme. Note, in the above-mentioned reaction, the precursor of the C-terminal .alpha.-amidated peptide as a substrate for a C-terminal .alpha.-amidating enzyme is a peptide or protein represented by a general formula X-R-Gly, wherein R represents an amino acid residue which is to be .alpha.-amidated at the C-terminal thereof, Gly represents a glycine residue, and X represents a remaining part of the peptide or protein.
Because of the importance of clarifying the mechanism of .alpha.-amide formation in tissues, and the promising usefulness of the enzyme for the production of C-terminal .alpha.-amidated peptides using, for example, recombinant DNA techniques, many attempts to purify the enzyme have been made but the enzyme has not so far been obtained in a pure state. In porcine pituitary, Bradburg, A. F. et al, Nature, 298, 686-688, 1982, first characterized the .alpha.-amidating activity of converting a synthetic substrate D-Tyr-Val-Gly to D-Tyr-Val-NH.sub.2, and demonstrated that the C-terminal glycine in the substrate serves as a nitrogen-donor for .alpha.-amidation. Eipper et al, Proc. Natl. Acad. Sci. US, 80, 5144-5148, 1983, reported that the .alpha.-amidating enzyme derived from the pituitary gland requires a copper cation and ascorbate for its activity. Husain, I. et al., FEBS Lett., 152 227-281, 1983; and Kizer, J. S. et al, Proc. Natl. Acad. Sci. US, 81, 3228-3232, 1984, also reported a C-terminal .alpha.-amidating enzyme, but did not report a purified enzyme. Recently, Murthy A. S. N. et al, J. Biol. Chem. 261, 1815-1822, 1986, partially purified a C-terminal .alpha.-amidating enzyme from the pituitary gland of cattle, and showed that several types of enzymes having different molecular weights and electric charges are present. Nevertheless, no type of enzyme has been homogeneously purified.
Recently, Mizuno et al. succeeded in isolating a C-terminal .alpha.-amidating enzyme in a homogeneous and pure form from a skin of Xenopus laevis; see Mizuno, K. et al, Biochem. Biophys. Res. Commun. 137, 984-991, 1988, and Japanese Patent Application No. 61-131089, and further succeeded in determining an entire primary amino acid sequence of the C-terminal .alpha.-amidating enzyme of a skin of Xenopus laevis origin by obtaining and sequencing cDNA; see Mizuno, K. et al, Biochem. Biophys. Res. Commun. 148, 546-552, 1987. Nevertheless, since proteins exhibiting a C-terminal .alpha.-amidating activity in mammalian tissues are present in a very small amount, are unstable, and are not homogeneous, it is very difficult to isolate and purify such proteins from the mammalian tissues, and no one has succeeded in this to date. Therefore, currently it is not clear whether there is one or more than one C-terminal .alpha.-amidating enzyme in a mammal, and if there is more than one enzyme, whether these enzymes have the same or a different substrate specificity.